SspA controls spore development by affecting septum placement
The SspA protein has been identified as an abundant protein in wild-type spores and is absent from the spores of a sigF null mutant. Bioinformatic analysis identified both an N-terminal signal sequence and a lipoprotein signature suggesting that SspA is exported through the cell membrane and attached to it, presumably from the outside. The rest of the SspA protein sequence comprises of two PepSY domains that are widespread in bacteria and fungi and found often in the propeptide domains of M4 proteases (Yeats et al., 2004). Where examined, the PepSY motif of the propeptide was shown to function as an intramolecular inhibitor preventing premature activation of the protease (Braun et al., 2000; Tang et al., 2003; Gao et al., 2010). While the M4 peptidases are well characterized, no clear biological role was established for those PepSY-family proteins that do not possess additional domains with enzymatic activity. The presence of the PepSY domain in this diverse family of secreted and cell wall-associated proteins suggested a regulatory role for protease activity in the local environment of the cell (Yeats et al., 2004). However, recent studies of PepSY-domain transmembrane proteins suggested that these domains can regulate the activity of proteins other than proteases. For example, the PepSY proteins BqsP and BqsQ of Pseudomonas aeruginosa are proposed to regulate the activity of a two-component system that senses extracellular Fe(II) (Kreamer et al., 2012). Similarly, the PepSY protein YycI regulates the YycFG two-component system in B. subtilis (Szurmant et al., 2007). Structural analysis proposed a common structural fold for the PepSY domain, the beta-lactamase inhibitor protein fold (BLIP) and two other protein folds, suggesting that all these protein folds function as inhibitors by binding a partner domain that is located either within the same protein or on a separate protein (Das et al., 2010).
Interestingly, one of the cortex-lytic enzymes in B. subtilis, SleB, is associated with YpeB, a protein with two copies of the PepSY domain (Boland et al., 2000). YpeB has been implicated in the control of a peptidoglycan amidase, SleB, important during spore germination (Atrih and Foster, 2001). YpeB was required for SleB localization (Chirakkal et al., 2002) and it was suggested that YpeB might recruit SleB to the spore cortex. However, recently the inhibition of SleB by YpeB was demonstrated, suggesting that the PepSY protein YpeB controls SleB activity (Li et al., 2013) perhaps by inhibiting the premature activation of the SleB lytic activity in the spores of B. subtilis.
SspA possesses two PepSY domains but lacks any catalytic peptidase domain, so its domain organization is very similar to that of YpeB. However, germination of the sspA mutant spores was not affected (data not shown). Instead, lack of SspA altered septum formation on all media tested and spore maturation when grown on minimal medium. Therefore, we hypothesize that SspA might control the activity of a specific peptidase or peptidoglycan hydrolase involved in either septum formation or spore maturation. Septum positioning depends on the key cell division protein FtsZ that assembles into 50–100 regularly spaced FtsZ rings marking future septum sites (Schwedock et al., 1997). In E. coli or B. subtilis, FtsZ positioning is regulated by FtsZ antagonist proteins. These include the MinCD complex located at cell poles promoting FtsZ ring formation at mid-cell and the chromosome associated SmlA or Noc proteins, which prevent FtsZ ring formation and therefore septum formation over the chromosomes (Adams and Errington, 2009; Lutkenhaus, 2012). In Streptomyces no apparent homologues of either the Min system or nucleoid occlusion proteins have thus far been identified. Instead, formation of the FtsZ rings is under a positive control by SsgB in Streptomyces (Willemse et al., 2011; Jakimowicz and van Wezel, 2012) while FtsZ protein levels are negatively affected by CrgA (Del Sol et al., 2006, Del Sol et al., 2003). Following on from the formation of the FtsZ rings, very little is known about the recruitment of specific enzymes for septum synthesis in Streptomyces. Interestingly, the FtsW–FtsI protein pair, which is established in septum formation in E. coli, was proposed for FtsZ ring stabilization in Streptomyces (Mistry et al., 2008; Bennett et al., 2009; McCormick, 2009). In addition, the SsgA-like proteins (SALPs) have been implicated in both septum positioning (SsgA and B) and in septum formation (SsgC-G) (Noens et al., 2005; Willemse et al., 2011; Jakimowicz and van Wezel, 2012).
The late sigma factor, SigF is associated with the control of spore maturation, which is a post-septation event. However, the sigF null mutant produces smaller spores than the wild-type (Potuckova et al., 1995). This could be the result of incorrect septum positioning and possibly altered FtsZ placement suggesting that SigF is active when septa are formed. Similarly, the uneven septum positioning in the sspA mutant could arise from altered FtsZ positioning. Alternatively, SspA might influence the recruitment of cell wall lytic or synthetic enzymes after FtsZ ring formation. Interestingly, monitoring SspA localization using an SspA–mCherry fusion confirmed that in addition to its presence in mature spores, SspA accumulated at sporulation septa (Fig. 5B). This suggests that SspA functions as early as septum formation. It will be important to establish the potential link between SspA and the Fts proteins (FtsZ, FtsW and FtsI), CrgA and the SALPs.
After the completion of septation, spore wall synthesis is governed by the cytoskeletal proteins MreB and Mbl (Mazza et al., 2006; Heichlinger et al., 2011). MreB polymers first assemble at sporulation septa, followed by spreading to the entire spore wall (Mazza et al., 2006). This pattern is reminiscent of SspA localization, raising the possibility that SspA positioning might be MreB-dependent. On the other hand, lack of SspA affects septation while MreB only controls post-septational events (Mazza et al., 2006) suggesting that SspA might function prior to MreB assembly during spore development. A recent search for MreB partner proteins established a complex interaction pattern among members of the proposed ‘Streptomyces spore wall synthesizing complex’, SSSC (Kleinschnitz et al., 2011). A knockout mutant of SCO2097, a putative membrane protein identified among the SSSC proteins, produced elongated spores with sensitivity to heat and cell wall-damaging agents (Kleinschnitz et al., 2011). Interestingly, these elongated spores resemble those of the sspA mutant; however, the latter did not exhibit sensitivity to lysozyme or heat (data not shown). In addition, spore wall hydrolytic enzymes have also been shown to affect spore shape (Haiser et al., 2009). It will be of interest to test whether any of the penicillin-binding proteins of the SSSC, including SCO3901, SCO3580 and FtsI (Kleinschnitz et al., 2011) or any of the hydrolytic enzymes, RpfA, SwlA, SwlB and SwlC (Haiser et al., 2009) are targets of or partnered by SspA.
Transcription of sspA is under the control of the sigma factor, SigF and the principal developmental regulator, BldD
Both the production of SspA protein and transcription of sspA are dependent on sigF in vivo, in S. coelicolor (Figs 1 and 4). Moreover, in vitro transcription assays (Fig. 4) confirmed that SigF is sufficient to initiate transcription from the sspA promoter. Hence, in this report we have presented the first example of a SigF target promoter, sspAP where transcription is initiated by RNA polymerase holoenzyme containing the sigma factor, SigF in the absence of any activator. SigF belongs to a group of nine RNA polymerase sigma factors (SigB, F, G, H, I, K, L, M and N) that control response to environmental stresses (SigB, H, I, L and M) or morphological differentiation (SigH, F and N) or in some cases both (SigH and SigB) in S. coelicolor (Potuckova et al., 1995; Cho et al., 2001; Kelemen et al., 2001; Sevcikova et al., 2001; Viollier et al., 2003; Lee et al., 2005). Members of this, so called, SigB-family of S. coelicolor resemble the general stress response sigma factor SigB of B. subtilis (see review, Price, 2000). Predictably, the putative promoter sequence of sspA (GTGT-16N-GGTTAC) resembles the consensus target sequence of SigB both in S. coelicolor and in B. subtilis (Fig. 4E; Petersohn et al., 1999; Price, 2000; Lee et al., 2004). Interestingly, the weak similarity between the two sigF-dependent promoters, sspA and whiEP2, together with the unusually long spacer between the −10 and −35 sequences of whiEP2 might explain why SigF was not sufficient to initiate transcription from whiEP2 in vitro (Kelemen et al., 1998). Identification of further SigF target promoters is paramount in order to establish a consensus SigF target sequence and to address the fundamental question of how members of the SigB-family with potentially overlapping promoter specificity can control distinct sets of genes in vivo. One such mechanism could include specific activators, as it was proposed for the promoters whiEP2 and nepA, which are putative targets for SigF and SigN respectively (Kelemen et al., 1998; Dalton et al., 2007).
Alternatively, transcriptional repressors could restrict expression of target genes both in time and in space, allowing limited access to promoter sites by cognate sigma factors. Interestingly, gel shift assays together with DNase I footprinting demonstrated a DNA binding activity from wild-type cell extracts collected at early stages of development, when sspA was not expressed, suggesting a putative repressor targeting sspA transcription. Both in vivo and in vitro experiments confirmed that this repressor is BldD, a key developmental regulator of Streptomyces morphogenesis. bldD was among the first developmental genes identified in the study of morphological differentiation in Streptomyces (Merrick, 1976). The bldD mutant fails to progress to aerial development and is also blocked in the production of several secondary metabolites (Elliot et al., 1998). BldD, a small DNA-binding protein, has been shown to target, and mainly repress, the transcription of developmental genes, such as the sigma factor genes, whiG, bldN and sigH, during early development while co-ordinating the timing and, in some cases, the location of their expression at later stages of differentiation (Elliot et al., 2001; Kelemen et al., 2001). Recently, the genome-wide BldD regulon has been extensively defined by chromatin immunoprecipitation-microarray analysis identifying ∼ 167 transcription units targeted by BldD (den Hengst et al., 2010). The location of the BldD binding site at the sspA promoter is consistent with BldD functioning as a repressor of sspA, perhaps by blocking access of other SigB-like sigma factors to the sspA promoter during early developmental stages. Surprisingly, microarray analysis showed that sspA transcription was not upregulated but absent in the bldD null mutant (den Hengst et al., 2010). This suggests that either the sspA promoter is recognized exclusively by SigF, which is naturally absent from the bldD mutant, or, more likely, no other SigB-like sigma factors capable of initiating sspA transcription were active under the conditions of the microarray analysis. Interestingly, one of the BldD targets identified in this analysis is the gene encoding SCO4677 (den Hengst et al., 2010), which has been demonstrated to bind SigF, potentially functioning as an anti-sigma factor (Kim et al., 2008). Thus BldD is linked to the sigF regulon via sspA, and perhaps also via SCO4677. Moreover, BldD has also been shown to target cell division genes (den Hengst et al., 2010), such as ftsZ, ssgA and ssgB or the smeA-ssfA operon encoding a DNA translocase required for correct chromosome segregation during sporulation (Ausmees et al., 2007). Hence, BldD appears to co-ordinate the expression of proteins that are required at the time of septum formation, including the expression of SspA.